Bardos, Julia Ildiko; (2001) Characterisation of HPC3, a new human polycomb group protein. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Polycomb Group (PcG) proteins are a conserved group of transcriptional repressors, mainly known for their role in stably maintaining the repressed state of homeotic and Hox genes, after their expression patterns have been established early in embryonic development. Thus, the Polycomb Group constitutes an important part of a cellular transcriptional memory system. Loss of PcG function leads to homeotic transformations in Drosophila and corresponding shifts in Hox gene expression in vertebrates. PcG proteins form multiprotein complexes of varying composition that associate with chromatin, and it has been postulated that they achieve gene silencing by altering higher order chromatin structure. But the exact mechanism by which PcG proteins establish repression is still unclear. A yeast two-hybrid screen with the PcG protein RING1 as bait led to the identification of a new protein that shows strong sequence similarity with other Polycomb homologues and was therefore termed human Polycomb 3 (HPC3). The hpc3 gene and its murine homologue are ubiquitously expressed in adult tissues, but in contrast to other polycomb genes, mpc3 is highly expressed during embryogenesis up to 8.5 days post coitum and becomes drastically downregulated at later stages, indicating a possible unique role ofpc3 early in development. Analysis of the protein-protein interactions showed that the HPC3 C-Box is necessary and sufficient for the interaction with RING1, but that this interaction depends only partially on the RING finger domain of RING1. HPC3 also interacts with the PcG protein BMI1 and colocalises with BMI1 and RING1 in distinct nuclear domains, called PcG bodies. Consistent with its role as a PcG member, HPC3 is able to act as a long-range transcriptional repressor when targeted to a reporter gene. Surprisingly, analysis of the different domains of HPC3 revealed that its conserved C-Box, which mediates the repression function of the other Pc homologues, is not the repression domain of HPC3. Instead, an internal part of the HPC3 protein mediates silencing of reporter gene activity. Transient transfection experiments show that the same domain is involved in mediating localisation of a HPC3-GFP fusion protein to distinct nuclear foci, indicating that it represents a functional domain of the HPC3 protein. These data suggest that HPC3 is a new member of the mammalian Polycomb Group in terms that it colocalises and interacts with other PcG proteins and is able to repress gene activity. At the same time, its expression profile suggests that it might have a unique role in embryogenesis. Another feature that distinguishes HPC3 from the other Pc homologues is that its conserved C-Box is not the domain responsible for the repression activity of HPC3, but that an internal part mediates transcriptional repression as well as localisation of the protein. Through interaction with other proteins, HPC3 potentially becomes involved in processes like cell proliferation, senescence, apoptosis and leukaemogenesis.

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